Bioactivity of Backhousia Citriodora: Antibacterial and Antifungal Activity

76 J. Agric. Food Chem. 2003, 51, 76−81

Bioactivity of Backhousia citriodora: Antibacterial and Antifungal Activity

JENNY M. WILKINSON,MICHAEL HIPWELL,TRACEY RYAN, AND HEATHER M. A. CAVANAGH*

School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia

Backhousia citriodora products are used as bushfoods and flavorings and by the aromatherapy industry. The antimicrobial activity of 4 samples of B. citriodora oil, leaf paste, commercial tea (0.2 and 0.02 g/mL), and hydrosol (aqueous distillate) were tested against 13 bacteria and 8 fungi. Little or no activity was found to be associated with the leaf tea and hydrosol, respectively. Leaf paste displayed antimicrobial activity against 7 bacteria including Clostridium perfringens, Pseudomonas aeruginosa, and a hospital isolate of methicillin resistant Staphylococcus aureus (MRSA). The 4 essential oils were found to be effective antibacterial and antifungal agents; however, variation was apparent between oils that did not correlate with citral content. The antimicrobial activity of B. citriodora essential oils was found to be greater than that of citral alone and often superior to Melaleuca alternifolia essential oil. B. citriodora has significant antimicrobial activity that has potential as an antiseptic or surface disinfectant or for inclusion in foods as a natural antimicrobial agent.

KEYWORDS: Backhousia citriodora; essential oil; antimicrobial; citral; lemon myrtle

INTRODUCTION confirmed the antimicrobial activity of the oil; however the number of bacteria tested was limited and only single samples Backhousia citriodora or lemon myrtle has been identified of oil were investigated (12-14). The antimicrobial actions of as one of the plants that are likely to undergo major commercial B. citriodora are believed to be directly related to the high citral exploitation over the next 5 years (1, 2). The leaves are used content; however, it is known that the antimicrobial activity of both as a bushfood and to produce an essential oil (3). The essential oils can differ from that of their major constituents, essential oil is then either incorporated into food products, probably due to synergistic or antagonistic effects of minor cosmetics, or toiletries or used as an aromatherapy oil. The components (15, 16). leaves contain between 1.1 and 3.2% oil and can be of one of two chemovars, the most common of which is the citral form In this study we investigate the antimicrobial activity of B. (4, 5). Citral, an isomeric mix of geranial and neral, is a known citriodora oil and hydrosol, both of which are used as antimicrobial agent with activity against both bacteria and fungi antimicrobial agents. As this plant is also commonly used as a (6, 7). For example, citral is thought to be responsible for the bush food, for example as a tea or included in cheeses, the resistance to postharvest fungal infection of lemons (8). Citral antimicrobial activity of leaf paste and leaf tea derived from B. present in essential oils may also control food-borne pathogens citriodora were also included. In addition, as previous unpub- and prevent spoilage (6). Essential oils with citral as a main lished data from this laboratory have indicated that variation in constituent include lemon, lemon verbena, and lemongrass. antimicrobial activity occurs in different B. citriodora oils, we - have included samples from four different sources. This report These oils are also known for their antimicrobial activity (9 s 11) although their citral content is considerably less than that presents the results of the first phase of this work determination of B. citriodora. of the antibacterial and antifungal properties of B. citriodora essential oils, leaf paste, tea, and hydrosol (aqueous distillate). The levels of citral in B. citriodora oil can vary between 80 and 98% with each of the other components (e.g. linalool and myrcene) accounting for less than 1% each (5). B. citriodora MATERIALS AND METHODS oil is reputed to possess antimicrobial activity; however, there All test substances were derived from Backhousia citriodora and has been little scientific investigation of the antibacterial or consisted of four essential oils, obtained from independent sources, a antifungal activity of this oil. Recent study into the activity of commercial brand of B. citriodora tea, a hydrosol, and crushed dried B. citriodora oil against a limited number of microorganisms leaf material. All assays were repeated a minimum of two times. Preparation of Test Material. All four 100% pure, steam-distilled, * Corresponding author. E-mail: [email protected]. Tel: +61 (2) B. citriodora essential oils were provided by primary producers and 6933 2501. Fax: +61 (2) 6933 2587. labeled A, B, C, or D, respectively, upon arrival. Citral content for

Table 1. Antibacterial and Antifungal Activity of Backhousia citriodora Essential Oils as Determined by the Disk Diffusion Assaya

Backhousia citriodora essential oil conc growth 100% 10 % 1% 0.1% Melaleuca alternifolia test organism properties conditions (°C) oil A oil B oil A oil B oil A oil B oil A oil B essential oil (100%) Alcaligenes faecalis G −ve 37 40.75 46 14.25 25.5 8.75 8.5 0 0 13 Clostridium perfringens G +ve 37 11.5 11 0 8.5 0 0 0 0 19 Echerichia coli G −ve 37 10 11 10.25 11.25 7.5 7.5 0 0 15 Echerichia coli type Bδ G −ve 37 nt nt 16. 5 18 9.25 8 0 0 20.5 Enterococcus faecalis G −ve 37 12.75 16 3.5 7.5 0 0 0 0 9 Mycobacterium phlei acid-fast 37 nt nt 7.5 12.25 0 0 0 0 20.5 MRS Aδ G +ve 37 11.5 16.5 7.75 0 0 0 0 0 18 Pseudomonas aeruginosa G −ve 37 0 0 0 0 0 0 0 0 0 Staphylococcus aureus G +ve 37 18 32 11. 5 13 0 0 0 0 12 Salmonella california G −ve 37 7.75 8.5 0 9.25 0 8 0 0 20 Salmonella enteritidis G −ve 37 10 9 7.5 9.75 0 8 0 0 19 Salmonella typhimurium G −ve 37 8 8 8.5 8.5 7 7 0 0 18.5 Shigella sonnei G −ve 37 15.75 0 18 33 8.25 8.5 0 0 15 Candida albincans yeast 37 0 0 0 (11.5) 0 (15) 0 0 0 0 0 Aspergillus niger fungi 37 >90* >90* >90* >90* >90* >90* 0 12 25.5 Fusarium graminearum fungi 30 >90* >90* 18 9.5 0 0 0 0 nt Leptospaeria maculans fungi 30 >90* >90* 21 13 0 0 0 0 nt Microsporum gypsium fungi 30 >90* >90* 25 0 0 0 0 0 55 Trichophyton mentagrophytes fungi 30 >90* >90* 40 16.5 0 0 0 0 nt Trichophyton rubrum fungi 30 >90* >90* 45 40 0 0 0 0 57.5 Trichophyton tonsurans fungi 30 >90* >90* 11 16 8 10 8 10 nt

a All figures indicate the mean of at least two assays. Unless otherwise indicated, all tests were performed on nutrient agar plates. Key: >90* indicates no growth on plates containing test substance; E. coli Bδ and MRSAδ are hospital isolates; nt indicates not tested; G −ve ) gram negative; G +ve ) gram positive. these oils was between 93 and 98% according to CG-MS data supplied deionized water (10-1-10-4) before adding 1.5 mL of each dilution to by each producer. All oils were stored in the dark at room temperature, a further 13.5 mL of molten agar, giving final concentrations of 10-2- and the date of breaking the seal was recorded. Melaleuca alternifolia 10-5. A 15 mL volume of agar containing the diluted test substance oil was donated by one of the primary producers. was subsequently placed in a sterile Petri dish and allowed to set. A A commercial brand of lemon myrtle leaf tea was obtained and loopful of each microorganism (1 × 106 organisms/mL), taken from a infused in boiling water for 20 min under foil to prevent vapor loss at fresh 24 h broth culture, was streaked onto a plate of each test substance two concentrations,5gin25mLofboiling water and5gin250mL at each concentration after the method of Mitscher et al. (17). The plates of boiling water. These represent a 10 and 1 times concentrate of the were then incubated at appropriate growth conditions and checked after manufacturers recommended concentration of tea. An additional sample 24 and 48 h. Microbial growth was scored on a scale of 1-4, where 0 of5gin25mLofwater cooled to 45 °C was also included. Infusions equals no growth and 4 equals full growth as determined by growth of were subsequently passed through a 0.25 µm syringe filter (Sartorius) organism on control plates. Control plates consisted of agar containing and used immediately. either 15 mL of nutrient agar alone, 10% sterile deionized water Dried leaf material was made into a paste in sterile, room-temperature (produces normal growth of all microorganisms), or commercial water (2:1 ratio), briefly (1 min) ground in a mortar and pestle, and antibiotics. Antibiotic controls consisted of either streptomycin sulfate used immediately. (final concentration 10 µg/mL) or amphotericin B (final concentration Citral (analytical grade, Sigma) was prepared at a concentration of 10 µg/mL). All organisms used in this study were susceptible to at 10% and 1% in sterile water in the presence or absence of 0.5% Tween least one of theses antibiotics with the exception of the hospital isolate 80 and used immediately. MRSA. All organisms within this study displayed expected antibiotic Microorganisms and Media. A variety of microorganisms (bacteria sensitivity profiles (data not shown). and fungi) that differ in cell structure, biochemical properties, and Disk Diffusion Assay. The 24 h broth cultures of bacteria were growth conditions were used in antimicrobial assays (listed in Table freshly prepared for each assay. Nutrient agar plates (15 mL) were 1). To ensure that the chemical composition of the growth media did prepared, allowed to set, and then surface dried (37 °C, 30 min). A 1 not interfere with the diffusion of the oil components, all organisms mL volume of bacterial broth culture (1 × 106 organisms/mL) was were grown on nutrient broth (10 g of tryptone,5gofyeast extract, subsequently poured evenly over the surface of the dried agar plates, 10 g of sodium chloride, water to 1 L) and/or nutrient agar (as for and the plates were placed at 37 °C for approximately 20 min until nutrient broth plus 15 g of agar/L of media). All assays were repeated, bacterial overlay had dried on the surface.A6mmsterile paper disk independently, a minimum of two times. All organisms were originally (Wattman) was placed onto the dried surface, in the center of the plate, obtained from the culture collection of the University of New South and 12 µL of test substance was gently placed onto the disk. Plates Wales with the exception of the hospital isolates of methicillin resistant were subsequently incubated at the appropriate temperature for 24 h. Staphylococcus aureus (MRSA) and Escherichia coli type B (E. coli Zones of inhibition were calculated by measuring diameter in mm type B). (including disk). Control plates consisted of nonimpregnated disks and Normal growth of all microorganisms was evaluated by growth on disks impregnated with 12 µL of sterile distilled water, both of which nutrient agar at appropriate conditions (37 or 30 °C as shown in Table resulted in full growth of all organisms. Disks impregnated with 12 1) in the absence of any test substance. Where possible, organisms µL of 100% tea tree oil (Melaleuca alternifolia) were also included were grown preferentially at 37 °C to avoid the effects of temperature for comparison. This assay was repeated for a limited number of variation on loss of volatile components from the oils. Anaerobic organisms (Table 3) with addition of 0.5% Tween 80 into the test conditions were achieved by placing agar plates in a “GasPak” anaerobic substance. All assays were repeated, independently, at least twice. jar (Sigma). Antimicrobial assays were carried out using standard agar dilution and disk diffusion methods. RESULTS Agar Dilution Assays. Dilutions of each test substance were prepared in cooled (42 °C) molten nutrient agar (1.5 mL of test B. citriodora hydrosol, leaf paste, and leaf tea were tested substance plus 13.5 mL of agar; 10-1 dilution) or prediluted in sterile against 15 microorganisms, Alcaligenes faecalis, Clostridium 78 J. Agric. Food Chem., Vol. 51, No. 1, 2003 Wilkinson et al. perfringens, Echerichia coli, Echerichia coli type B, Entero- Table 2. Determination of the Antimicrobial Activity of Backhousia coccus faecalis, Mycobacterium phlei, MRSA, Pseudomonas Citriodora Oil by Agar Dilutiona aeruginosa, Staphylococcus aureus, Salmonella california, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, dilution of oil in agar Candida albincans, and Aspergillus niger. With the exception test organism 10% 1% 0.1% 0.01% 0.001% of the tea prepared at 10 times recommended strength (0.2 Staphylococcus aureus 013 4 4 g/mL), no antimicrobial activity was detected at any concentra- Echerichia coli 014 4 4 tion for either the hydrosol or the tea infusions irrespective of Salmonella typhimurium 023 4 4 assay system used (data not shown). The tea (0.2 g/mL) showed Mycobacterium phlei 013 4 4 Clostridium perfringens 001 4 4 a slight inhibition of Clostridium perfringens witha9mmzone Candida albicans 012 4 4 of inhibition (including disk). The leaf paste, however, dem- Aspergillus niger 000 2 4 onstrated antimicrobial activity against 7 bacteria; A. faecalis (13 mm), C. perfringens (29.5 mm), M. phlei (15 mm), MRSA a Key: 0 ) no growth of organism; 1 ) 25% of normal growth; 2 ) 50% of (16 mm), P. aeruginosa (13 mm), S. aureus (16 mm), and C. normal growth; 4 ) 100% of normal growth as compared to water controls. All albicans (8 mm). M. alternifolia oil (100%) demonstrated figures are based on the mean of at least two independent assays. All assays activity against all organisms tested except P. aeruginosa (Table were carried out on nutrient agar. Results shown are for B. citriodora oil A. 1). Initially, two B. citriodora essential oils, A and B, were Tween 80. The addition of 0.5% Tween 80 into the disk evaluated using the disk diffusion assay. With the exception of diffusion assay had variable effects (Table 3). The presence of P. aeruginosa, both oils demonstrated varying degrees of Tween 80 significantly reduced the susceptibility of both E. coli antimicrobial activity against all 21 organisms tested, with the and S. aureus to 10% citral and S. aureus to 1% citral while A. fungi being the most susceptible (Table 1). Both oils demon- faecalis became more susceptible to 1% citral in the presence strated activity against six of the organisms (A. faecalis, E. coli, of Tween 80. Similarly, the apparent susceptibility of E. coli E. coli type B, S. typhimurium, S. sonnei, A. niger, and T. and S. typhimurium to B. citriodora oil B (10%) and oil D tonsurans) at a concentration of 1%. Oil B also demonstrated (10%), respectively, was also reduced. Conversely the presence antimicrobial activity against S. enteritidis and S. california at of 0.5% Tween 80 apparently increased the susceptibility of S. a 1% concentration. Oil B continued to demonstrate activity aureus to B. citriodora oils C (10% and 1%) and D (10%), S. against A. niger, and both oils inhibited growth of T. tonsurans typhimurium susceptibility to oil B (10%), and A. faecalis to at a concentration of 0.1%. Variable results were, however, oil B (1%). No organism tested displayed any growth, irrespec- apparent with selected organisms. For example, E. faecalis tive of oil source, when 10% B. citriodora essential oil was repeatedly displayed no susceptibility to either oil at concentra- incorporated into the agar (Table 3). No organism displayed tions of 100% yet demonstrated susceptibility at both 10% and any growth inhibition in the presence of 0.5% Tween 80 alone 1% concentrations. Results for C. albicans were also variable. (data not shown). Further repeats of the agar dilution assay for C. albicans results showed that, of the three assays carried out, 10% concentrations of all oils and commercial citral resulted no inhibition at oil concentrations of 100% or 1% was observed, in no growth of any organism tested (Table 3). yet one of three assays at a 10% concentration indicated an 11.5 In general, the antimicrobial activity of B. citriodora oil was mm (oil A) and 15 mm (oil B) zone of inhibition while the greater than that of citral against all organisms, although remaining two assays again indicated no activity. variation in the efficacy of the different oils was apparent. Oils Although both oils demonstrated activity against the majority A and B were more effective than citral against all five of the organisms, there was considerable variation in the activity organisms tested. Citral was more effective against E. coli than of oil A compared to oil B. For example, C. perfringens was oils C and D and also against S. aureus than oil C. All four oils apparently more susceptible to oil B than oil A while MRSA were more effective than citral against S. typhimurium, C. albicans, and A. faecalis (Table 3). was more susceptible to oil A than oil B at concentrations of 10%. M. altenifolia oil (100%) displayed varying degrees of The antimicrobial activity of B. citriodora oil was greater activity against all organisms tested, with the exception of P. than that of Melaleuca alternifolia oil against A. faecalis, M. aeruginosa. phlei, S. aureus, F. graminearum, M. gypsium, T. mentagrophytes, and T. rubrum. The activity of M. alternifolia oil (100%) A limited number of organisms, chosen on the basis of their was significantly greater than that of B. citriodora against C. varying susceptibilities to B. citriodora oil, were utilized to assay perfringens and the Salmonella spp. tested. All other organisms antimicrobial activity of B. citriodora oil A via agar dilution, were equally susceptible to the two types of essential oil. S. aureus, E. coli, S. typhimurium, M. phlei, C. perfringens, C. albicans, and A. niger. Antimicrobial activity was demonstrated against all organisms at dilutions up to 1% with some inhibition DISCUSSION of all bacteria other than E. coli still evident at 0.1% (Table 2). The antimicrobial activity of B. citriodora tea was investigated A. niger and Cl. perfringens growth was completely inhibited on the basis of three tea preparations: tea prepared as per at 1%. Growth of all organisms was normal at dilutions greater manufacturers instructions in boiling water, tea prepared as per than 0.1% with the exception of the fungi A. niger which was manufacturers instruction but in water at 45 °C to prevent loss fully inhibited by dilutions of 0.01% and demonstrated ap- of any antimicrobial activity through evaporation, and finally proximately 50% inhibition at a concentration of 0.001% B. tea prepared at 10 times the manufacturers recommended citriodora oil. concentration. Only the 10× concentrate tea displayed any The effect of adding a solubilizing agent, Tween 80, into the antimicrobial activity against the 15 organisms tested. A small disk diffusion assay was also undertaken as part of a comparison zone of inhibition (9 mm) was consistently detected when this to the antibacterial activity of B. citriodora oil and commercial tea preparation was tested against Clostridium perfringens. Like citral. The disk diffusion assay was repeated, with a limited the 10× concentrate tea, C. perfringens was also susceptible to number of organisms, in the presence and absence of 0.5% the leaf paste but consistently produced a much greater zone of Bioactivity of Backhousia citriodora J. Agric. Food Chem., Vol. 51, No. 1, 2003 79

Table 3. Antimicrobial Activity of Backhousia Citriodora Oil and Citral in the Presence and Absence of 0.5% Tween 80a

0.5% Tween 80 test organism sample assay method conc of oil (%) incorporated? E. coli S. aureus S. typhimurium C. albicans A. faecalis B. citriodora oil A agar incorporation 10 − ng ng ng ng ng disk diffusion 10 − 10.25 11.5 8.5 22 14.5 disk diffusion 10 + 8 13 0 22 19 disk diffusion 1 − 7.5 7 7 0 8.75 disk diffusion 1 + 07 0 8 7 B. citriodora oil B agar incorporation 10 − ng ng ng ng ng disk diffusion 10 − 11.25 13 8.5 17 25.5 disk diffusion 10 + 0 11 9 20 14 disk diffusion 1 − 7.5 7 7 7 8.5 disk diffusion 1 + 07 0 8 8 B. citriodora oil C agar incorporation 10 − ng ng ng ng ng disk diffusion 10 − 0 4.5 6.5 22 25 disk diffusion 10 + 0 8.75 8 20 28 disk diffusion 1 − 0 0.5 0 7 9 disk diffusion 1 + 0 2.5 0 8 9 B. citriodora oil D agar incorporation 10 − ng ng ng ng ng disk diffusion 10 − 08 7 1627 disk diffusion 10 + 0 15 0 18 32 disk diffusion 1 − 07 0 8 8 disk diffusion 1 + 07 0 109 citral (Sigma) agar incorporation 10 − ng ng ng ng ng disk diffusion 10 − 17 11 0 0 15 disk diffusion 10 + 7.5 4.25 0 0 15 disk diffusion 1 − 0 6.5 0 0 0 disk diffusion 1 + 0 0.25 0 0 0

a Key: growth of all organisms in disk diffusion assay was measured in diameter of zone of including the disk (6 mm). All figures are based on the mean of at least two independent assays. Growth on agar dilution was recorded as no growth (ng) for each of the organisms in this table. All assays were carried out on nutrient agar. inhibition (29.5 mm). This is probably due to the much greater concentrations higher than 0.01%. The results for the disk concentration of leaf material present in the leaf paste and diffusion assay did however show some degree of incon- consequent higher concentration of oil. Interestingly, of the sistency with certain microorganisms, most notably E. coli B, seven organisms inhibited by the leaf paste, only M. phlei was Salmonella sp., and C. albicans. C. albicans has previously been more sensitive to 100% M. alternifolia oil. In addition, P. reported to be susceptible to B. citriodora oil with a minimum aeruginosa growth was inhibited by the B. citriodora leaf paste inhibitory concentration (MIC) of 0.03% (14). This incon- but not the teas, hydrosols, or any of the essential oils used in sistency may be explained by the use of oils from different this study including M. alternifolia. These findings are con- sources (and hence slight variation in the chemical composition sistent with previous reports which show an innate resistance of the oils) or different growth conditions for the organisms or of P. aeruginosa to M. alternifolia oil, although it has been by the use of different isolates of C. albicans. Variation in strain suggested that some strain variation in susceptibility may occur susceptibility of microorganisms to essential oils has previously as reports of P. aeruginosa being susceptible to M. alternifolia been reported (18). This phenomenon warrants further investiga- oil have appeared in the literature (14, 18). No antimicrobial tion. activity was detected in the B. citriodora hydrosol tested in this While the standard methods of disk diffusion and agar dilution study. utilized in this study are suitable for the aqueous teas and Two methods were trialed to determine the antimicrobial hydrosols, the insoluble nature of the majority of constituents activity of B. citriodora essential oil, the agar dilution assay of essential oils often limits their diffusion through agar, leading and the disk diffusion assay. The agar dilution assay was initially to incorrect or inconsistent results (18). Many studies now use used to evaluate antimicrobial activity against a limited number solubilizing agents, such as Tween 80, to overcome this problem of microorganisms. Using this assay, some degree of anti- (11, 18). The introduction of 0.5% Tween 80 into the disk microbial activity was demonstrated by B. citriodora oil against diffusion assay produced variable effects. With some organisms all organisms to a 1% concentration. Growth of all the bacteria the presence of Tween 80 significantly reduced the susceptibil- was normal at dilutions greater than 0.1%, except that growth ity; with others the susceptibility was increased, while others of Aspergillus niger was prevented up to dilutions of 0.01% were unaffected. No organism displayed any growth inhibition with growth at 0.001% approximately half that on the control in the presence of 0.5% Tween 80 alone. While some authors plate. The results for the agar dilution assay were consistent continue to publish data relating to the antimicrobial activity and reproducible, and the oil was found to form a stable mix of essential oils on the basis of the disk diffusion assay (with with the agar. or without solubilizing agents), from this study we would In the disk diffusion assay, the oil was found to prevent the conclude that this assay did not yield consistent results and is growth of all organisms tested at 100% concentration other than not recommended for future testing of essential oils. Conversely, P. aeruginosa, E. coli B, and C. albicans. At this concentration the agar dilution method gave consistent reproducible results B. citriodora oil was consistently more effective than 100% M. for both the oils and citral. alternifolia oil against the bacteria A. faecalis, E. faecalis, S. While this study found that the antimicrobial activity of B. aureus, and the fungi Aspergillus, Microsporum, and Tricho- citriodora was, in general, greater than that of citral alone, Hayes phyton. B. citridora was found to be particularly effective against and Markovic (14) reported little difference between the the fungi with the growth of some being inhibited to at all antimicrobial activity of citral and B. citriodora oil. The different 80 J. Agric. Food Chem., Vol. 51, No. 1, 2003 Wilkinson et al. oils used in this present study did show surprising degrees of for preservation and prevention of microbial spoilage. Further variation with oils A and B being consistently more effective studies are currently underway to investigate the biocidal versus than oils C and D despite similar citral content. The fact that the biostatic effect of this oil, as determination of the biocidal the antimicrobial activity of B. citriodora oil was greater than activity will play a vital role in the potential use of this oil as that of citral against all organisms tested other than E. coli raises a therapeutic agent. Evaluation of the effect of B. citriodora interesting questions and suggests that other compounds within oil on other bacteria and fungi of importance to the medical, the oil effect the susceptibility of the microorganisms. food, agricultural, and horticultural industries in addition to Hayes and Markovic (14) also report that B. citriodora oil is determination of any antiviral, antiparasitic, or antioxidant more effective than M. alternifolia oil against S. aureus, E. coli, activity is currently under investigation. P. aeruginosa, C. albicans, A. niger, and Klebsiella pneumoniae. ACKNOWLEDGMENT The B. citriodora oils utilized in this study also displayed greater activity than M. alternifolia oil against a wide range of The authors thank the following companies for the kind donation organisms, although not against P. aeruginosa or E. coli. of the Backhousia citriodora products used in this study: Toona Inhibition of P. aeruginosa was only found with the B. Essential Oils; Australian Botanical Products; Summit Coffee; citriodora leaf paste in this study. Again, strain related or growth Australian Native Foods Management. We also thank Mr. R. related differences might be responsible for these differences. Johnston of Wagga Wagga Base Hospital for supplying the P. aeruginosa was grown on NA in this study while Iso-sensitest MRSA and E. coli B isolates. broth (Oxoid) was used by Hayes and Markovic (14). Various LITERATURE CITED degrees of P. aeruginosa susceptibility to M. alternifolia oil have been reported (14, 19). We have also found that while the (1) Dyer, K. Eat Well Conference, Adelaide; http://www.chdf.org.au/ type strain E. coli described in the present investigation was eatwellsa/ conference/15.html (accessed 8/04/00), pp 1-29. not more susceptible to B. citriodora oil than M. alternifolia (2) Fergus, J. What will be the next big oil from Australia? oil, other strains of E. coli, such as the hospital isolate of E. International Conference on Essential Oils and Aromas, Hong coli type B, are more susceptible. K. pneumoniae was not Kong. investigated in this study. (3) Hegarty, M. P.; Hegarty, E. E.; Wills, R. B. H. Food safety of Australian plant bushfoods; Rural Industries Research & De- Few other studies on B. citriodora oil have been reported.B. velopment Corp.: Kingston, ACT, Australia, 2001. citriodora oil is reported to be nonmutagenic; however, only (4) Brophy, J.; Goldsack, R.; Fookes, C.; Forester, P. Leaf oils of concentrations of 10% and below were tested (12). Its use as a the genus Backhousia (Myrtaceae). J. Essent. Oil Res. 1995, 7, bushfood would support the likelihood that the oil is non- 237-254. mutagenic at lower concentrations. There is some conflicting (5) Southwell, I. A.; Russell, M.; Smith, R. L.; Archer, D. W. evidence relating to the mammalian cell toxicity, in vitro, of B. Backhousia citriodora F. Muell. (Myrtaceae), a superior source - citriodora oil, however, with one report stating no toxicity was of citral. J. Essent. Oil Res. 2000, 12, 735 741. detected against HeLa and Hep2 cell lines at concentrations less (6) Kim, J.; Marshall, M. R.; Wei, C. Antibacterial activity of some essential oil components against five foodborne pathogens. J. than 1% (12). In contrast, a second report states an inhibitory Agric. Food Chem. 1995, 43, 2839-2845. concentration of 50% of between 0.04% and 0.01%, although, (7) Inouye, S.; Tsuruoka, T.; Uchida, K.; Yamaguchi, H. Effect of again, this may reflect the different cell lines used in each study sealing and Tween 80 on the antifungal susceptibility testing of (Hep2 and HeLa vs F1-73, skin fibroblasts, and HepG2) (14). essential oils. Microbiol. Immunol. 2001, 43, 201-208. Certain cell lines, like HepG2, are known to be more sensitive (8) Rodov, V.; Ben-Yehoshua, S.; Fang, D. Q.; Kim, J. J.; Ashkenazi to essential oils (20). From the limited data available to date, Preformed antifungal compounds of lemon fruit: citral and its no anticancer activity has been associated with B. citriodora relation to disease resistance. J. Agric. Food Chem. 1995, 43, oil in that no growth inhibition was detected against colon, 1057-1061. breast, leukemia, and salivary gland (cell line) carcinomas in (9) Inouye, S.; Takizawa, T.; Yamaguchi, H. Antibacterial activity vitro (12). On the basis of case studies, the oil is reported to of essential oils and their major constituents against respiratory tract pathogens by gaseous contact. J. Antimicrob. Chemother. have antiherpesvirus activity (21). 2001, 47, 565-573. The results of this study show that B. citrodora oil is an (10) Wannissorn, B.; Jarikasem, S.; Soontorntanasart, T. Antifungal effective antibacterial and excellent antifungal agent that displays activity of lemon grass oil and lemon grass cream. Phytother. no apparent difference in the susceptibility of gram negative Res. 1996, 10, 551-554. and gram positive organisms. Slight differences were found in (11) Hammer, K. A.; Carson, C. F.; Riley, T. V. Antimicrobial activity the antimicrobial activity of the different B. citriodora essential of essential oils and other plant extracts. J. Appl. Microbiol. 1999, - oils utilized in this study, all of which were obtained from 86, 985 990. V difference sources (A-D), and only the leaf paste was found (12) Ryan, T. Antimicrobial acti ity of natural medicines; Summer scholarship report; Charles Sturt University: Wagga Wagga, to have any activity against P. aeruginosa. The differences Australia, 2000. between oils may be due to different levels of citral or other (13) Ryan, T.; Cavanagh, H. M. A.; Wilkinson, J. M. Antimicrobial components, growth conditions, or distillation procedures. Not activity of Backhousia citriodora oil. Simply Essent. 2000, 38, only is the composition of essential oils and plant extracts known 6-8. to vary according to local climatic and environmental conditions (14) Hayes, A. J.; Markovic, B. Toxicity of australian essential oil but distillation times and temperatures can also significantly Backhousia citriodora (Lemon myrtle). Part 1. Antimicrobial affect the oil constituents (22). activity and in vitro cytotoxicity. Food Chem. Toxicol. 2002, - The data generated, however, would indicate that B. citriodora 40, 535 543. (15) Lis-Balchin, M.; Deans, S. G.; Eaglesham, E. Relationship essential oil has potential as an antiseptic or surface disinfectant, between bioactivity and chemical composition of commercial although further research is needed to determine whether there essential oils. FlaVour Fragrance J. 1998, 13,98-104. are other biological activities of this oil that may be of benefit. (16) Lis-Balchin, M.; Deans, S. G. Studies on the potential usage of For example, B. citriodora oil is also a powerful antifungal agent mixtures of plant essential oils as synergistic antibacterial agents and its antimicrobial activity may have role in the food industry in foods. Phytother. Res. 1998, 12, 472-475. Bioactivity of Backhousia citriodora J. Agric. Food Chem., Vol. 51, No. 1, 2003 81

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